TaqMan Real-time Reverse transcription-PCR Assays Research Articles

Comparison of three TaqMan real-time reverse transcription-PCR assays in detecting SARS-CoV-2

Quick and accurate detection of SARS-CoV-2 is critical for COVID-19 control. Dozens of real-time reverse transcription PCR (qRT-PCR) assays have been developed to meet the urgent need of COVID-19 control. However, methodological comparisons among the developed qRT-PCR assays are limited. In the present study, we evaluated the sensitivity, specificity, amplification efficiency, and linear detection ranges of three qRT-PCR assays, including the assays developed by our group (IPBCAMS), and the assays recommended by WHO and China CDC (CCDC). The three qRT-PCR assays exhibited similar sensitivities, with the limit of detection (LoD) at about 10 copies per reaction (except the ORF 1b gene assay in CCDC assays with a LoD at about 100 copies per reaction). No cross reaction with other respiratory viruses were observed in all of the three qRT-PCR assays. Wide linear detection ranges from 106 to 101 copies per reaction and acceptable reproducibility were obtained. By using 25 clinical specimens, the N gene assay of IPBCAMS assays and CCDC assays performed better (with detection rates of 92 % and 100 %, respectively) than that of the WHO assays (with a detection rate of 60 %), and the ORF 1b gene assay in IPBCAMS assays performed better (with a detection rate of 64 %) than those of the WHO assays and the CCDC assays (with detection rates of 48 % and 20 %, respectively). In conclusion, the N gene assays of CCDC assays and IPBCAMS assays and the ORF 1b gene assay of IPBCAMS assays were recommended for qRT-PCR screening of SARS-CoV-2.

Development of two TaqMan real-time reverse transcription-PCR assays for the detection of severe acute respiratory syndrome coronavirus-2

The outbreak of coronavirus disease 2019 (COVID-19) in Wuhan, China, was caused by a novel coronavirus (CoV), named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The rapid detection of viral nucleic acids is critical for the early identification of infected cases. We have developed two TaqMan real-time reverse transcription-PCR assays to detect SARS-CoV-2. The designed primers target the nucleocapsid (N) and open reading frame (ORF) 1b gene regions, where the probes discriminate SARS-CoV-2 from other human and animal CoVs. The sensitivities are one genomic copy per reaction for the N gene assay and ten copies for the ORF 1b gene assay. The overall linear detection ranges are 1–106 and 10–106 copies per reaction for the N gene assay and the ORF 1b gene assay, respectively. Surveillance of 23 suspected COVID-19 patients demonstrated that SARS-CoV-2 could be detected from 100% (23/23) and 62.5% (16/23) of clinical specimens by the N gene assay and the ORF 1b gene assay, respectively. All of the samples not detected by the ORF 1b gene assay were throat swabs, indicating a lower viral load in the upper respiratory tract and the relatively lower sensitivity of the ORF 1b gene assay. The assays developed in the present study offer alternative diagnostic tests for COVID-19.

Design and Validation of an H5 TaqMan Real-Time One-Step Reverse Transcription-PCR and Confirmatory Assays for Diagnosis and Verification of Influenza A Virus H5 Infections in Humans

Increasing diversity among influenza H5N1 viruses has resulted in the need for sensitive and specific diagnostic assays, fully validated for the detection of H5 viruses belonging to all hemagglutinin (HA) clades, particularly the recently circulating H5N1 viruses of clade 2. In this report, the development and validation of a real-time, one-step TaqMan reverse transcription-PCR (RT-PCR) assay specific for the detection of influenza A H5 viruses from clades 1, 1', 2, and 3 is described. The real-time assay for H5 virus was shown to be highly sensitive, detecting H5 virus levels of <1 PFU from each of the HA clades. Specificity of the H5 RT-PCR for influenza A H5 viruses was demonstrated by using influenza A viruses of different subtypes, clinical samples containing influenza A viruses H1N1, H3N2, and H5N1, influenza B viruses, and other respiratory viruses. The usefulness of the inclusion of a distinguishable assay positive control and of confirmatory assays for the laboratory diagnosis and verification of H5 virus infections was demonstrated. A real-time RT-PCR pyrosequencing assay, a restriction enzyme digestion assay, and direct sequencing of the H5 real-time RT-PCR amplicon were validated for the confirmation of H5 detection by the diagnostic real-time assay. The H5 real-time assay was applied to diagnostic testing for suspected cases of influenza A virus H5 infection in the United Kingdom. Influenza A H5 viruses were not detected in the cases analyzed; however, influenza A H3N2 virus was detected in 57% of the suspected cases of H5. The H5 TaqMan real-time RT-PCR and confirmatory assays will be useful tools for the laboratory surveillance and rapid diagnosis of H5 infections in humans.

Global differential gene expression in response to growth temperature alteration in group A Streptococcus.

Pathogens are exposed to different temperatures during an infection cycle and must regulate gene expression accordingly. However, the extent to which virulent bacteria alter gene expression in response to temperatures encountered in the host is unknown. Group A Streptococcus (GAS) is a human-specific pathogen that is responsible for illnesses ranging from superficial skin infections and pharyngitis to severe invasive infections such as necrotizing fasciitis and streptococcal toxic shock syndrome. GAS survives and multiplies at different temperatures during human infection. DNA microarray analysis was used to investigate the influence of temperature on global gene expression in a serotype M1 strain grown to exponential phase at 29 degrees C and 37 degrees C. Approximately 9% of genes were differentially expressed by at least 1.5-fold at 29 degrees C relative to 37 degrees C, including genes encoding transporter proteins, proteins involved in iron homeostasis, transcriptional regulators, phage-associated proteins, and proteins with no known homologue. Relatively few known virulence genes were differentially expressed at this threshold. However, transcription of 28 genes encoding proteins with predicted secretion signal sequences was altered, indicating that growth temperature substantially influences the extracellular proteome. TaqMan real-time reverse transcription-PCR assays confirmed the microarray data. We also discovered that transcription of genes encoding hemolysins, and proteins with inferred roles in iron regulation, transport, and homeostasis, was influenced by growth at 40 degrees C. Thus, GAS profoundly alters gene expression in response to temperature. The data delineate the spectrum of temperature-regulated gene expression in an important human pathogen and provide many unforeseen lines of pathogenesis investigation.